This invention relates to a method for concentrating and recovering microorganisms from difficult-to-separate samples, particularly environmental samples, for the purpose of their analysis or identification.
Existing protocols for detection of microorganisms present in natural environments are, in general, cumbersome, time consuming, or inadequate with respect to sensitivity or accuracy. Traditionally, environmental samples are processed to recover and cultivate targeted organisms. These organisms are then screened to verify identity. Cultivation methods usually require extensive incubation periods and a variety of tests may be needed to identify targeted organisms. Antibody-based detection systems, which were developed in the 1970s, are rapid but suffer from the possible generation of false positive signals. While DNA-based detection systems developed in the 1980s are accurate and rapid, their use for detection is limited by interferences imposed by substances present in environmental samples. Consequently, DNA-based detection requires sample treatment to ensure reliable detection of microorganisms. Reported methods for processing environmental samples to allow DNA-based detection of microorganisms typically involve release of DNA from the microorganisms, while still suspended in the sample, and subsequent isolation and purification of the released DNA. These methods require many steps, are time consuming, employ caustic and toxic chemicals, generate low yields of DNA, and expose the released DNA to environmental substances which may adversely affect DNA integrity.
Environmental samples such as soil, sewage, and surface water are complex mixtures which are heterogeneous in both their solid and soluble components. In composition, the samples sometimes contain high concentrations of solid sediments, colloids, emulsions, soluble and insoluble salts, biopolymers, biodegraded debris, inert materials and contaminating industrial and natural chemicals. The heterogeneity and complexity of environmental samples can prevent or invalidate direct detection methods. Microorganisms can adhere to particulates, rendering the microorganisms inaccessible to detection by conventional approaches which rely on liquid filtrate samples. Also chemicals and solutes contained within the samples can interfere or inhibit test methods. Sample coloration, for example, can obstruct test results when colors are part of the assay.
Detection of microorganisms and/or their products is complicated by the fact that in many instances, sample materials contain only small numbers of microorganisms (10.sup.2 to 10.sup.5 cells/gm). Further, in many cases the microorganisms do not comprise a single strain or even a single genus but are a diverse collection of many different types of organisms. For these reasons, detection requires highly sensitive methods possessing broad spectrum specificity or a means of selecting specific organisms. Because of the requirements of both high sensitivity and broad spectrum specificity, detection approaches such as immunoassays directed toward the cells or their products are subject to chemical interferences.
To circumvent these problems, some manner of sample treatment has been typically required to concentrate the small numbers of organisms present in field sample materials and to free them from interfering materials which prevent analysis or cause inaccurate or false results. Generally, treatment processes can involve centrifugation, membrane filtration or chemical precipitation steps.
Solution gradients or density gradients are utilized in biochemical research to separate macromolecules such as proteins, DNA and RNA, and larger aggregates such as viruses and cells.
Solution gradients usually utilize a solute of varying concentrations to aid in the separation of particles. Examples of appropriate solutes are: sucrose, CsCl, Percoll.TM. (colloidal silica coated with polyvinyl pyrrolidone), ficoll (a copolymer of sucrose and epichlorohydrin), metrizamide (3-acetylamino-5-N-methyl-acetylamino-2,4,6-triiodobenzoyl-glucosamine), Nycodenz.TM., sodium acetate, glycerol and mixtures thereof. Particles are separated either by their velocity of sedimentation in a centrifugal field, or by their density in a centrifugal field if there is an isopycnic point within the solution column in the tube. Faster, or denser particles will appear lower in the tube.
After the sample has been subjected to centrifugation, the particles are recovered for analysis. Fractionation methods and apparatus used to recover the sample in the gradient may involve the transfer of the entire gradient or certain layers or bands of the solution gradient to other vessels. It is often desired to extract only desired bands from the solution gradient for electron microscopy, liquid scintillation or gel electrophoresis.
The Membrane Filter (MF) method utilizes micropore filters through which samples are passed so that the bacteria are retained on the surface of the filter. This method is often used when bacterial populations are very small, and a large sample is needed to get adequate numbers. The filter is then placed on the surface of a chosen medium, incubated, and the bacterial colonies growing on the membrane filter surface are counted and evaluated. This method is widely used and provides good results when combined with proper reagents and media.
It is an object of the present invention to provide an improved method to collect and recover microorganisms from environmental samples.
Other objects and advantages of the present invention will be apparent to those skilled in the art.